CA2871736A1 - Quantitation of biomarkers for the detection of prostate cancer - Google Patents

Abstract

The present invention provides biomarkers that are useful in determining whether a subject has cancer, specifically prostate cancer. Specifically, betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine can be used to diagnose an individual with prostate cancer, one at risk for developing prostate cancer, and/or to determine the prognosis of a subject with prostate cancer. This invention also relates to multiplexed assays for quantitating such biomarkers.

Description

QUANTITATION OF BIOMARKERS FOR THE DETECTION OF PROSTATE
CANCER
RELATED APPLICATIONS
100011 This application claims the benefit under 35 U.S.C. 119(e) of U.S.
Provisional Application Nos. 61/639,768 filed on April 27, 2012, 61/764,288 filed on February 13, 2013, and 61/772,226 filed on March 4, 2013, which are hereby incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
100021 This invention relates to novel biomarkers for treating, diagnosing, and preventing prostate cancer. The invention also relates to methods of identifying, characterizing, and using such prostate cancer biomarkers. This invention also relates to multiplexed assays for quantitating biomarkers.
BACKGROUND
100031 Prostate cancer is one of the most common malignancies in the US
(1). It is clinically heterogeneous, with a highly variable natural history (2). The discovery and widespread utilization of serum prostate specific antigen (PSA) monitoring for early detection has greatly changed the way prostate cancer is diagnosed and treated. However, PSA lacks specificity as a screening tool for prostate cancer, and there is really no lower limit of PSA that entirely excludes cancer (3). Thus, clinical decision making in prostate cancer places a significant burden upon biopsy results. Ultrasound guided needle biopsy is the standard for diagnosis however, a negative result does not exclude the presence of cancer.
Both sampling and analytical variables account for false negative results. In practice, false negative results engender a need for repeat biopsies which can delay diagnosis and treatment or unnecessarily subject cancer-free men to repeat biopsies and their attendant anxiety and risk (4, 5). The heterogeneity of prostate cancer is also a significant problem and while the incidence is high, the death rates from prostate cancer are relatively low as compared to those from the other major cancers such as lung, pancreas, and colon. The Gleason grading system which has been widely adopted for prostate cancer is a predictor of outcome (6). However, a major limitation of this grading system, and a result of aggressive screening procedures, is that a majority of newly diagnosed prostate cancer cases are Gleason score 6 or 7 tumors. These moderately differentiated tumors can either be indolent or aggressive (7).
100041 A.s referenced above, a determination of the prognosis of prostate cancer is guided by the Gleason grading system. In this system, a biopsy of the prostate tissue is harvested, fixed in formalin, embedded in paraffin, and then sliced and stained for viewing. The pathologist will then give the particular sample of tissue a grade or pattern based on the appearance of the tissue. The grade will range from 1 to 5, with a higher number indicating a more aggressive cancer. The pathologist gives a grade to the most common tumor pattern and then a grade to the second most common tumor pattern. These grades are added to provide the overall Gleason score. The Gleason score ranges from 2 to 10, with 10 having the worst prognosis.
100051 The Gleason score is only one component of prostate cancer staging.
The most common method of prostate cancer staging is promulgated by the American joint Committee on Cancer and is known as the wINM" system. There are two types of staging, the clinical stage and the pathologic stage. The clinical stage is determined prior to treatment such as surgical prostatectomy, and includes five key elements:
= The extent of the primary tumor (T category) = Whether the cancer has spread to nearby lymph nodes (N category) = The absence or presence of distant metastasis (M category) = The PSA. level at the time of diagnosis = The Gleason score, based on the prostate biopsy (or surgery) T describes the size of the primary tumor, N describes whether nearby lymph nodes are involved in the cancer and M describes metastasis or spread of the cancer.
00061 Following surgical prostatectomy, the prostate is carefully examined for assignment of pathologic stage. The "T" scale for prostate cancer is as follows:
Ti: tumor present, but not detectable clinically or with imaging T 1 a: tumor was incidentally found in less than 5% of prostate tissue resected (for other reasons) Tib: tumor was incidentally found in greater than 5% of prostate tissue resected Tic: tumor was found in a needle biopsy performed due to an elevated serum PSA
T2: the tumor can be felt (palpated) on examination, but has not spread outside the prostate T2a: the tumor is in half or less than half of one of the prostate gland's two lobes T2b: the tumor is in more than half of one lobe, but not both 12c: the tumor is in both lobes but within the prostafic capsule T3: the tumor has spread through the prostatic capsule (if it is only part-way through, it is still T2) T3a: the tumor has spread through the capsule on one or both sides T3b: the tumor has invaded one or both seminal vesicles T4: the tumor has invaded other nearby structures This ranking, coupled with the N and M, is combined with the histological assessment from the Gleason score to determine whether definitive treatment for the cancer should be taken or watchful waiting should be chosen.
100071 A variety of nomograms are available to assess the risk of aggressive prostate cancer including the d'Amico system (8). This assigns the following risk scores: Low-risk:
PSA less than or equal to 10, Gleason score less than or equal to 6, and clinical stage T1-2a;
intermediate risk: PSA between 10 and 20, Gleason score 7, or clinical stage T2b; High-risk:
PSA more than 20, Gleason score equal or larger than 8, or clinical stage T2c-3a. Definitive treatment entails radiation therapy, or prostatectomy. Therapy may also be deferred in an attempt to balance expected life span, the likelihood of treatment side effects, and quality of life. Watchful waiting (periodic clinical visits and PSA measurements) or active surveillance (periodic clinic visits and PSA measurements combined with scheduled repeat biopsies) are used when patients are comfortable with postponement of definitive therapy.
100081 The current methods for diagnosing and making prognostic decision-making for prostate cancer have limitations in that interobserver variability occurs, especially in the setting of small tumors. This is where quantitative information would be of value to patient and physician. Therefore, new quantitative methods to assist clinicians and pathologists in both diagnostic and prognostic decision making are needed to aid in the detection and treatment of prostate cancer.
100091 The majority of men with prostate cancer will die with their disease rather than of it (67), and there is a strong argument that screening has increased the detection of indolent tumors (68). Unfortunately, we lack clinical tools to distinguish indolent from aggressive prostate cancer (69), and it is estimated that over 1400 men need to be screened and nearly 50 men treated to prevent one prostate cancer death (70). Prostate cancers with similar microscopic features have variable clinical outcomes, reflecting genetic and biological variables of individual patients not recognized by microscopy alone (71).
Unfortunately, tissue architecture is often destroyed by extraction methods required for detection of molecular tissue biomarkers. Therefore, emerging methods for biomarker discovery and validation compete with histology for the same tissue are needed. This is especially so when small needle biopsies are utilized, which is the standard of care for diagnosis of suspected prostate cancer.

100101 Pathologic examination remains a gold standard for diagnosis, classification, and staging of tumors. This requires intact tissue while implementation of molecular tests often requires extraction methods that disrupt tissue. Metabolomics is a newer area of biospecimen analysis in which small molecules (--2kD) (e.g., metabolites), present in a biological sample, are extracted, detected and measured. The method has been employed in the study of the biochemical basis and mechanisms for diverse biological processes such as cancer diagnosis and monitoring progression, drug mechanism of action, drug toxicity, industrial bio-processing, etc.
100111 In order to analyze the metabolites of a biological sample, they must be extracted completely from the sample. Existing methods of biological extraction involve destroying the sample such that it can no longer be used for other analysis (e.g., histology). Fixation of tissue samples is usually done with formalin (formaldehyde in water), followed by histologic processing and sectioning, and this is the usual work flow that produces a slide for microscopic examination by a pathologist. The drawback of this method is that formaldehyde is not an effective extractant for metabolites. Therefore, under commonly known biological extraction methods, in order to conduct histology analysis and perform metabolomics, two tissue samples are required, one for each analysis. The current state of the art is that the second biopsy would be used only for metabolomics and would not be examined histologically. Thus, it cannot be known with certainty whether the biopsy used for metabolomics contained diseased tissue.
100121 Methods and reagents for performing metabolomics and subsequent histology on various tissue samples are described in detail in PCT Appl. No.

(W02011/146683), which is herein incorporated by reference in its entirety. A
preferred embodiment of that method involves contacting a single biological sample (e.g.
a tissue biopsy) with a solvent (e.g. ethanol or methanol) such that the extracted biochemical can be analyzed and the extracted tissue retains its cellular architecture so that it can be subsequently analyzed using standard histological methods (including cytological analysis). Using this method, a number of tissues have been extracted, their biological chemicals analyzed, and subsequent histology preformed. See, Shuster et al. "Molecular preservation by extraction and fixation, inPREF: a method for small molecule biomarker analysis and histology on exactly the same tissue." BMC Clinical Pathology 2011, 11:14, herein incorporated by reference in its entirety.
100131 The term, molecular preservation by extraction and fixation ("mPREF"), refers to a process of preserving cellular structure in tissue or cell specimens whilst extracting small molecules by immersing the tissue in a solution containing an organic solvent, then subsequently processing the exact same portion of tissue using histological methods. mPREF
enables both quantitation of biochemicals, including small molecule metabolites, and histological examination of the same tissue sample. mPREF permits quantitation of metabolites and histology in exactly the same tissue. This diminishes the competition of new molecular testing methods with standard histology for small amounts of tumor containing biopsy tissue. mPREF treated tissues can be used for all existing methods that use paraffin embedded tissue. The aqueous alcohol in mPREF selectively extracts small molecules from tissue, leaving macromolecules such as proteins, RNA, and DNA in place.
Existing powerful in-situ methods for detecting proteins (immunohistochemistry, IHC) and RNA and DNA
(fluorescence in situ hybridization, FISH) in intact tissue can continue to be used in mPREF
processed tissue.
100141 A biomarker is an organic biomolecule, the presence of which in a sample is used to determine the phenotypic status of the subject (e.g., cancer patient v. normal patient or prognosis of cancer patient). In order for the biomarker to be biologically relevant it should be differentially present in a sample taken from a subject of one phenotypic status (e.g., having a disease) as compared with another phenotypic status (e.g., not having the disease). Biomarkers, alone or in combination, provide measures of relative risk that a subject belongs to one phenotypic status or another. Therefore, they are useful as markers for disease (diagnostics), prognosis (i.e., state of the disease), therapeutic effectiveness of a drug (theranostics), drug toxicity, and predicting and identifying the immune response.
100151 There remains a need for multiplex assays to quantitate diagnostic cancer metabolites that are isolated from biological samples in a manner that allows such samples to be further analyzed using standard histological methods. The metabolites that are identified and characterized can then be used as cancer biomarkers.
100161 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
BRIEF SUMMARY OF THE INVENTION

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100171 In one embodiment, the invention provides a method for screening for prostate cancer in a subject by: (a) providing a biological sample from a subject; (b) detecting at least one biomarker in said sample, said biomarker selected from the group consisting of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine; and (c) correlating said detection with a status of prostate cancer or no prostate cancer.
100181 In a further embodiment, the invention provides a method for screening for prostate cancer in a subject by: (a) providing a biological sample from a subject; (b) detecting at least one biomarker in said sample, said biomarker selected from the group consisting of betaine, malate, proline, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine;
and (c) correlating said detection with a status of prostate cancer or no prostate cancer.
100191 In a further embodiment the detecting at least one biomarker is performed by mass spectrometry.
100201 In a still further embodiment, the biological sample is selected from the group consisting of biological fluid and tissue.
100211 In a further embodiment, the biological fluid is whole blood, serum, plasma, or urine.
100221 In a further embodiment, the tissue is a prostate tissue sample.
100231 In a further embodiment, the biological sample is contacted with a solvent capable of extracting the at least one biomarker.
100241 In a further embodiment, the solvent is methanol or ethanol.
100251 In another embodiment, the invention provides a method of diagnosing prostate cancer in a subject by: (a) obtaining one or more test samples from a subject;
(b) detecting at least one biomarker in the one or more test samples, wherein the biomarker is selected from:
betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine; (c) quantitating the amount of the at least one biomarker;
and (d) correlating the quantitation of the at least one biomarker with a diagnosis of prostate cancer.
100261 In yet another embodiment, the invention provides a method of diagnosing prostate cancer in a subject by: (a) obtaining one or more test samples from a subject;
(b) detecting at least one biomarker in the one or more test samples, wherein the biomarker is selected from:
betaine, malate, prolinc, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine; (c) quantitating the amount of the at least one biomarker; and (d) correlating the qua3ntitation of the at least one biomarker with a diagnosis of prostate cancer, wherein the correlation takes into account the amount of the at least one biomarker in the one or more test samples compared to a control amount of the at least one biomarker.
100271 In a further embodiment, the correlation takes into account the amount of the at least one biomarker in the one or more test samples compared to a control amount of the at least one biomarker.
100281 In a further embodiment, the test sample is selected from the group consisting of urine, whole blood, serum, plasma, and prostate tissue.
100291 In another embodiment, the invention provides a method of monitoring the effect of a prostate cancer drug or therapy on a subject by: (a) providing a biological sample from the subject; (b) contacting the biological sample with a solvent capable of extracting at least one prostate cancer biomarker selected from the group consisting of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosarnine; (c) quantitating the amount of the at least one prostate cancer biomarker; (d) providing the subject with an anti-prostate cancer drug or therapy; (e) quantitating the amount of the at least one prostate cancer biomarker using steps (a) and (h); and (f) corrlating the two measurements with a diagnosis that the prostate cancer is regressing or progressing.
100301 In another embodiment, the invention provides a multiplexed assay for screening for prostate cancer in a subject by: (a) providing a biological sample from a subject; (b) quantitating at least two or more biomarkers in said sample, said biomarkers selected from the group consisting of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine; (c) correlating said quantitation with a status of prostate cancer or no prostate cancer.
100311 In a further embodiment, the quantitating at least two or more biomarkers is performed by liquid chromatography in tandem with mass spectrometry.
100321 In a further embodiment, the biological sample is selected from the group consisting of biological fluid and tissue.

-7.-100331 In a further embodiment, the biological fluid is whole blood, serum, plasma, or urine.
100341 In a further embodiment, the tissue is a prostate tissue sample.
100351 In a further embodiment, the biomarkers betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine are quantitated in the same assay.
100361 In a further embodiment, the biomarkers betaine, malate, proline, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine are quantitated in the same assay.
100371 In another embodiment, the invention provides a multiplexed method for detecting prostate cancer in a subject by: (a) providing a biological sample from the subject; (b) contacting the biological sample with a solvent capable of extracting two or more prostate cancer biomarkers selected from the group consisting of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine;
(c) quantitating the amount of the two or more biomarkers present in the biological sample; and (d) correlating the amount of the two or more biomarkers with the presence or absence of prostate cancer.
100381 In a further embodiment, the quantitating differentiates between different stages of prostate cancer.
100391 In a further embodiment, the quantitating is part of a diagnosis or prognosis of prostate cancer in the subject.
100401 In a further embodiment, the solvent is methanol or ethanol.
100411 In a further embodiment, the step of performing additional histological analysis on the extracted biological sample.
100421 In another embodiment, the invention provides a method of diagnosing prostate cancer in a subject by: (a) obtaining one or more test samples from a subject;
(b) detecting at least one biomarker in the one or more test samples, wherein the biomarker is selected from the group consisting of betaine, malate, proline, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine; (c) quantitating the amount of the at least one biomarker;
(d) determining the Gleason score of the one or more test samples; (e) correlating the quantitation of the at least one biomarker and the Gleason score with a relative risk of T2 versus T3 prostate cancer.

100431 In another embodiment, the invention provides a kit for diagnosing prostate cancer in a subject with (a) a vial for collecting a biological sample from the subject; (b) a solvent for extacfing biomarkers from the biological sample, the biomarkers selected from the group consisting of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine; (c) instructions for performing the extraction of the biomarkers; (d) instructions for quantitating one or more of the biomarkers; (f) instructions for correlating the quantitation of the one or more biomarkers to a diagnosis of prostate cancer or normal.
BRIEF DESCRIPTION OF THE DRAWINGS
100441 The following figures are provided for the purpose of illustration only and are not intended to be limiting.
100451 FIGURE 1: A quantitation curve of uracil.
100461 FIGURE 2: A quantitation curve of N-acetylaspartate.
100471 FIGURE 3: A quantitation curve of xanthine.
100481 FIGURE 4: A quantitation curve of alanine.
100491 FIGURE 5: A quantitation curve of proline.
100501 FIGURE 6: A quantitation curve of betaine.
100511 FIGURE 7: A quantitation curve of cysteine.
100521 FIGURE 8: A quantitation curve of malate.
100531 FIGURE 9: Quantitation results of targeted biomarker compounds are provided in Figure 9.
100541 FIGURE 10: Figure 10 shows the concentration ranges where the measured values of the biomarkers of the present invention fell on the concentration standard curves. These are shaded gray.
100551 FIGURE 11: Figure 11 shows the actual values from the 29 prostate samples (15 tumor and 14 non-tumor) that were analyzed by the current method.
100561 FIGURE 12: Figure 12 shows the concentration ranges where the measured values of the biomarkers of the present invention fell on the concentration standard curves. These are shaded gray.
100571 FIGURE 13: A quantitation curve of uracil.
100581 FIGURE 14: A quantitation curve of N-acetylaspartate.
100591 FIGURE 15: A quantitation curve of xanthine.
100601 FIGURE 16: A quantitation curve of alanine.
100611 FIGURE 17: A quantitation curve of proline.

100621 FIGURE 18: A quantitation curve of betaine.
100631 FIGURE 19: A quantitation curve of cysteine.
100641 FIGURE 20: A quantitation curve of malate.
100651 FIGURE 21: A quantitation curve of N-acetylglucosamine.
100661 FIGURE 22: A graphical flow chart that represents the process of performing the extraction and metabolomics as described by the current invention and the subsequent histology of the tissue samples.
100671 FIGURE 23: A graph showing the difference between the concentration of the biomarkers uracil, N-acetylaspartate, proline, xanthine, betaine, malate, and N-acetylglucosamine in non-tumor tissue as compared to tumor tissue.
100681 FIGURE 24: A graph showing the difference between the concentration of the biomarkers alanine and cysteine in non-tumor tissue as compared to tumor tissue.
DETAILED DESCRIPTION OF THE INVENTION
100691 The invention is directed to biomarkers for prostate cancer. The invention is also directed to methods of detecting the presence of one or more biomarkers in order to make a diagnosis or prognosis of prostate cancer. The measurement of these markers, alone or in combination, in patient samples, provides information that the diagnostician can correlate with a diagnosis of prostate cancer, risk of developing prostate cancer, and/or prognosis of a subject with prostate cancer. In some embodiments, the biomarkers are betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine.
All nine of these biomarkers can be measured and quantitated at the same time in a single multiplex assay, which could provide very valuable information to the clinician or pathologist.
The assay can be varied to quantitate a smaller number of biomarkers if desired.
[0070] In one embodiment of the invention, mPREF was used to demonstrate that a subset of metabolites can be quantitated in 18 gauge core needle biopsies of prostate tissue. These metabolites can be used as clinically useful biomarkers. In another embodiment, the quantitation of these biomarkers can be used in the diagnosis or prognosis of prostate cancer.
Methods of the present invention can be used independently or in conjunction with currently accepted (or later developed) methods for diagnosing prostate cancer and/or determining the prognosis of a subject with prostate cancer. For example, methods of the present invention may be combined with histology methods.
100711 One aspect of this invention is an assay for quantitating select candidate diagnostic metabolites from cancer needle biopsy extracts using ultra-performance liquid chromatography coupled to a tandem mass spectrometry system (UPLC-MS/MS). In one embodiment, the cancer is prostate cancer. A subset of metabolites from prostate needle biopsies taken from surgical prostatectomy specimens prepared using molecular preservation by extraction and fixation ("mPREF") were identified as candidate prostate cancer diagnostic biomarkers. (66) In order to determine which metabolites would be the most useful, they were ranked based on the analytical technique that would be required for quantitation (e.g. GC/MS, LC/MS, etc.); the types of verification studies that could be used to confirm that the metabolite was a biomarker for cancer; and the likely ability that the metabolite could be used in a diagnostic assays. Based on this analysis, nine metabolites were identified as potential biomarkers for prostate cancer. In one aspect of the invention, UPLC-MS/MS can be used as the assay platform.
However, any LC/MS configuration can be used.
100721 The assay of the present invention was developed and used to quantitate the following biomarkers: betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine in 29 human prostate biopsy extracts. These involve 8 biochemical groups, which represent pathways of alanine and aspartate metabolism;
cysteine, methionine, SAM, and taurine metabolism.; glycine, serine, and threonine metabolism; urea cycle, arginine and proline metabolism; aminosugars, glycolysis, pentose metabolism, Krebs cycle, purine (hypoxanthine/inosine containing) and pyiimidine metabolism, respectively.
100731 These biomarkers can be used in a multiplexed assay for aiding in the prognosis and diagnosis of cancers, for example prostate cancer. One aspect of the present invention allows for the assay of all nine identified metabolites in a single LC-MS run in a quantitative manner on 18 gauge core needle biopsies. This allows for immediate application in the clinical setting.
Then, the tissue can be encased in paraffin and subjected to further processing and histology.
A flow chart outlining this procedure is shown in Figure 22.
100741 One advantage of the current invention is that the biomarkers can.
be quantitated at the time of a prostate biopsy rather than on prostate tissue samples procured from prostatectomy specimens. Prostatectomy results when the patient and physician have already made a decision to undergo definitive therapy, and have chosen radical prostatectomy.
Prognostic information derived from a prostatectomy specimen is not without value, however, the greater value resides in prognostic information contained in the prostate biopsy. The decision point prior to definitive therapy is more crucial, and this is when the patient has been diagnosed with prostate cancer following a biopsy. Prognostic markers useful at this point must therefore be applied to biopsy tissue. The assays of the present invention allow biomarkers to be quantitated from prostate biopsies, and, therefore, can provide information prior to definitive therapy. The treatment options available to patients now include watchful waiting and active surveillance. Active surveillance protocols are problematical since the decision to undergo definitive therapy is substantially influenced by Gleason grading. As described above, this has limitations as interobserver variability occurs.
Therefore, quantitative information would be of value to patient and physician.
100751 Quantitation of the biomarkers at the time of prostate biopsy rather than on prostate tissue samples procured from prostatectomy specimens may also be advantageous because it could reduce the effects of ischemia time on metabolites. Metabolite data on human prostate tissue has utilized cryopreserved tissue obtained from prostatectomy specimens. These may be subject to warm ischemia (intraoperafive) times of at least 40-60 minutes, prior to any time involved with specimen transport and processing.
100761 Another advantage of the current invention is that it allows for the sampling of the entire prostate when implemented in vivo. Biomarkers can be quantitated in each core regardless of whether histologic tumor is present, and multiple cores with tumor can be sampled. The capability to broadly sample the prostate could be very important since prostate cancer can be heterogeneous.
100771 A fluffier benefit of this invention is that the analysis can be performed on a smaller amount of tissue than the existing diagnostic/prognostic methods.
Specifically, this method can be performed on a single 18 gauge needle biopsy that only removes ¨ 5 mg of tissue. Previous methods for tissue biopsy have required large tissue removal (1 gram or greater) or multiple biopsies of smaller volume (e.g. 18 gauge core biopsies that harvest about 5 mg of tissue). The problem with these two biopsy methods is that they require a significant quantity of tissue to be removed causing greater discomfort and trauma to the subject.
100781 In the methods of the present invention, the fixation of the biomarkers can be performed on a tissue sample and that same tissue sample can then be sent on for further histological evaluation. This is an improvement over the traditional methods of extraction, in which tissue samples were fixed in formalin since formalin is not a suitable extractant for metabolites. Formalin is ineffective in extracting the metabolites and it is reactive so it can alter the metabolite chemistry. Therefore two separate samples (composed of different tissues) had to be harvested using the traditional methods. In the methods of the present invention, a single biological sample (e.g. a tissue biopsy) is contacted with a solvent (e.g. ethanol or methanol) such that the extracted metabolite can be analyzed and the extracted tissue retains its cellular architecture so that it can be subsequently analyzed using standard histological methods (including cytological analysis). The metabolites can then be quantitated and biomarkers may be identified.
100791 A further aspect of this invention is for the analysis to be a multiplexed assay. A
multiplex assay is an assay that simultaneously measures multiple biomarkers in a particular sample. The biomarkers can be measured directly from a patient sample with minimal preparation. This allows for a real time assessment of the patient's health in the clinical setting as it relates to the state of the disease. For example, in one embodiment of the invention, samples of prostate cancer biopsy were extracted from various cancer patients.
The metabolites were extracted and a series of biomarkers were discovered (betaine, malate, proline. N-acetylaspartate, uracil, xanthine, cysteine. N-acetylglucosamine, and alanine).
These biomarkers can be used in a multiplexed analysis of that patient's disease state.
100801 Another advantage of the present invention is that it provides a high throughput method for analyzing biomarkers. Alternatively, using intact tissue, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are used to measure biomarkers in intact tissue. IFIC and FISH are technically challenging, generally performed one at a time, and require microscopic interpretation, introducing inter-observer variability.
IHC still requires optical detection and interpretation.
100811 Another advantage of the present invention is that the results are quantitative.
Metabolite measurements as described herein can be expressed as absolute molar amounts of metabolites. This is a key distinction with IHC results which are notoriously difficult to quantitate.
100821 Betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine as biomarkers for prostate cancer, as well as methods and uses thereof, are disclosed. These biomarkers are overexpressed in patients with cancer, specifically prostate cancer. These biomarkers can, therefore, be utilized to diagnose patients with cancer, or to diagnose patients at risk for developing cancer. In some embodiments, the invention provides a method of diagnosing prostate cancer in a subject, comprising detecting the differential expression of at least one biomarker in the one or more test samples obtained from the subject, wherein the biomarker is betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine. In another embodiment, the invention provides a method of determining the prognosis of a subject with prostate cancer, comprising detecting the differential expression of at least one biomarker in the one or more test samples obtained from the subject, wherein the biomarker is betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine.
100831 In one embodiment of the present invention, a method of diagnosing cancer, specifically prostate cancer, or risk for developing cancer in a subject is provided. This method comprises the steps of (a) providing a biological sample from the subject; (b) contacting the biological sample with an extraction reagent capable of extracting the cancer biomarkers comprising betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine; (c) determining the amount of the biomarkers; and (d) correlating the amount biomakers to a prostate cancer diagnosis.
100841 The amount of cancer biomarkers (i.e., betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine) in normal biological samples can be assessed in a variety of ways as described herein. In one embodiment, the normal or control amount of biomarkers can be determined by assessing the amount of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine, in one or more samples obtained from one or more individuals without cancer.
100851 Using the methods of the invention, levels of prostate cancer biomarkers (i.e., betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine) are determined in a biological sample from a subject suspected of having prostate cancer and in one or more comparable biological samples from normal or healthy subjects (i.e., control samples). A level of prostate cancer biomarker (i.e., betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine) detected in a biological sample from a subject suspected of having prostate cancer that is higher than the prostate cancer biomarker level detected in a comparable biological sample from a normal or healthy subject, indicates that the subject suspected of having prostate cancer has or is likely to have prostate cancer.
100861 The biomarkers of the present invention can also be quantitated and correlated with various stages of a disease. For example, the biomarkers can be used to determine whether a subject has stage pT2 disease or pT3 disease of prostate cancer. At present, there is no way of confidently distinguishing pT2 from pT3 disease unless a prostatectomy is performed. Using the methods of the invention, levels of prostate cancer biomarkers (i.e., betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine) could be determined in a biological sample from a subject suspected of having prostate cancer and in one or more comparable biological samples from subjects with different stages (i.e., pT2 or pT3) of the diease. A level of prostate cancer biomarker (i.e., betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine) detected in a biological sample from a subject suspected of having prostate cancer that is comparable to prostate cancer biomarker level detected in a comparable biological sample from a subject with a pT2 or pT3 stage of the disease, would be instructive as to what stage of prostate cancer is present in the tested subject.
100871 In other aspects of the invention, the quantitation of biomarkers in conjunction with mPREF techniques can be done for biomarkers other than those for prostate cancer. The quantitation methods of the current invention are applicable to any tissue biopsy and any disease state, and are not limited to a single organ site. Other possible applications of the methods of the present invention include inflammatory skin diseases, diabetes, allografts for analysis of rejection, muscle and nerve biopsies, and cytologic specimens such as fine needle aspirates and smears.
100881 In accordance with the invention, at least one biomarker may be detected. It is to be understood, and is described herein, that one or more biomarkers may be detected and subsequently analyzed, including several or all of the biomarkers identified.
100891 It is to be understood that normal histology methods can also be used in conjunction with the methods of the invention. This is accomplished by imaging the slides to estimate tumor volume. The methods may combine automated feature recognition with manual (pathologist-assisted) feature recognition and assignment to optimize workflow. One aspect of the invention is to produce a high throughput system that can reasonably approximate the surface areas including 1) Total surface area of specimen, 2) Surface area of benign epithelium, 3) Surface area of tumor epithelium, and 4) Surface area of stroma. This information can then be used with computer-assisted software to efficiently correlate metabolite data with histology.
100901 The methods of the invention can also be used in conjunction with a graphical user interface ("GUI") that displays normalized metabolite values with standard text based pathology biopsy reports in a visually ergonomic fashion. Specifically, the invention can be used to display a pathological report that displays relative risk with each positive core on the "front sheet" readily visible to the clinician (urologist; oncologist) end user. A more detailed display with quantification would be on a "back sheet". Each specimen received (each core) in the laboratory requires generation of a pathology report which conveys in two or three lines of text, the diagnosis, Gleason score and grade, an estimate of the percent of biopsy involved by tumor, and the number of cores involved by tumor. The normalized metabolite data can be combined with the traditional pathology report. It can be read quickly, readily interpreted, and can be expressed as a relative risk of T3 vs. T2 disease etc. Recent studies have shown that many methods of digital image processing can be used for prostate imaging (63, 64, 65). These include computer-assisted tools and software for processing pathological prostate images for automatic classification and accurate grading of prostate tissues.
100911 Although mPREF techniques are described herein, the biomarkers of the present invention may be detected from any biological sample from a subject. The biological sample may be a biological fluid such as whole blood or serum. The biological sample may also be from tissue such as prostate tissue. Other examples of tissue specimen useful to practice the general methods of the present invention include samples taken from the prostate, central nervous system, bone, breast tissue, renal tissue, endometrium, head/neck, gall bladder, parotid tissue, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, urethra, liver, spleen, pancreas, thyroid tissue, heart, lung, bladder, adipose tissue, lymph node tissue, adrenal tissue, testis tissue, tonsils, and thymus.
100921 Biornarkers of the present invention may also be detected from biological fluid such as whole blood, serum, plasma, urine, tears, mucus ascites fluid, oral fluid, saliva, semen, seminal fluid, mucus, stool, sputum, cerebrospinal fluid, bone marrow, lymph, and fetal fluid.
The biological fluid samples may include cells, proteins, or membrane extracts of cells.
100931 "Subject" includes living and dead organisms. Examples of subjects include inainnials, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgertic nonhuman animals. Most preferably the subject is a human.
100941 The biomarkers of this invention can be isolated and purified from biological fluids, such as urine or serum. They can be isolated by any method known in the art, based on their mass, their binding characteristics and their identity as betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine.
For example, a biological sample comprising the biomarkers can be subject to chromatographic fractionation and subject to further separation.
100951 "Purified" means substantially pure and refers to biomarkers that are substantially free of other proteins, lipids, carbohydrates or other materials with which they are naturally associated.
Monitoring the Effect of an Anti-Prostate Cancer Drug or Therapy Administered to a Subject with Prostate Cancer 100961 In another embodiment of the present invention, a subject's prostate cancer status is determined as part of monitoring the effect of an anti-prostate cancer drug or a therapy administered to the subject diagnosed with prostate cancer. The effect of an anti-prostate cancer drug or a therapy administered to a subject with prostate cancer may include the worsening or improvement of prostate cancer processes.
100971 Using the methods of the invention, levels of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, or N-acetylglucosamine are determined in a biological sample from a subject at various times of having been given an anti-prostate cancer drug or a therapy. A prostate cancer biomarker level detected in a biological sample from a subject at a first time (ti; e.g., before giving an anti-prostate cancer drug or a therapy) that is higher than the prostate cancer biomarker level detected in a comparable biological sample from the same subject taken at a second time (t2; e.g., after giving an anti-prostate cancer drug or therapy), indicates that the cancer in the subject is regressing. Likewise, a higher prostate cancer biomarker level at a second time compared to a prostate cancer biomarker level at a first time, indicates that the cancer in the subject is progressing.
100981 In another embodiment, this method involves measuring one or more prostate cancer biomarkers, one of which may be betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, or N-acetylglucosamine, in a subject at least at two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The effect of the anti-prostate cancer drug or therapy on the progression or regression of the cancer is determined based on these comparisons. Thus, this method is useful for determining the response to treatment. If a treatment is effective, then the prostate cancer biomarker will trend toward normal, while if treatment is ineffective, the prostate cancer biomarker will trend toward disease indications.
100991 In another embodiment, the method involves measuring one or more metastases of prostate cancer biomarkers, one of which may be betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, or N-acetylglucosamine, in a subject at least at two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any.
This is done to assess the state of the disease, the progression of the disease and the likelihood of response to a treatment.
Kits for Diagnosing and Assessing Prognosis of Prostate Cancer 101001 The methods, alone or in combination of the present invention, may be provided in the form of a kit. Kits may further comprise appropriate controls and/or detection reagents. In an embodiment, the kit may include tools and reagents for the analysis of a tissue sample or biopsy. The kit may comprise a sample collection element and a tool for placing the biopsy or tissue sample into the collection element. The collection element may contain extraction solvent, a tool to retrieve the tissue following incubation, and a tool to place the collected tissue sample into a collection receptacle for histological analyses. For example, the kit may comprise a sample collection element, an extraction solvent, a tissue retrieval element, a retrieved tissue collection receptacle, sample labels, sample barcodes, and instruction protocol.
The instruction protocol may be provided as a printed form or booklet or on an electronic medium, such as, for example, a computer disk or other computer readable medium.
EXAMPLES
101011 The following examples are presented for the purpose of illustration only and are not intended to be limiting.
101021 Methods for quantitating potential prostate cancer biomarkers are provided below.
Materials and Methods 101031 Reference standards and stable isotope-labeled standards were purchased from Sigma-Aldrich (St. Louis, MO) including betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, and alanine. Stable isotope- labeled chemicals including betaine-trimethyl-d5 hydrochloride, xanthine 1,3-N15, cysteine C13, malate-2,3,3-d3, and L-proline 2,5,5-d3 were obtained from Cambridge Isotope Laboratories Inc. (Andover, MA) and uracil-d4, N-acetyl-aspartate 2,3,3-d3, and alanine-C13 were from CID/N ISOTOPES INC.
(Quebec, Canada). Chemicals including methanol (Optima LC-MS), acetonitiile (Optima LC-MS), and formic acid (Optima LC-MS) were purchased from Thermo Fisher Scientific (FairLawn, NJ).
Ammonium acetate and acetic acid (Glacial) were purchased from Sigma-Aldrich.
Sodium formate solution (0.05 M NaOH + 0.5% formic acid in 90:10 2-propanol: water, Waters Corp., Milford, MA) was used for instrument tuning and calibration. Ultrapure water was produced by Mill-Q Reference system equipped with a LC-MS Pak filter (Millipore, Billerica, MA).
I. Quan t it at ion Experiments 101041 A total of 29 extract samples were obtained from surgical prostatectomy specimens of patients who elected prostatectomy as a primary treatment. The biopsies were obtained ex vivo and were stored at 4 C until sample preparation and analysis. A pooled quality control sample was prepared by mixing each aliquot of the test samples for assay validation and concentration estimation.
Sample preparation 101051 Each 1 mL of sample was transferred to a 5-mL glass vial and dried under gentle nitrogen at room temperature (Glas-Col Nitrogen Evaporator System, Terre Haute, IN). The residue was reconstituted with 100 'IL of acetonitrile plus 100 IA, of water.
The mixture was centrifuged at 14,500 rpm for 20 min (Microfuge 22R centrifuge, Beckman Coulter, Inc., Atlanta, GA). A. 150-p.L aliquot of the resulting supernatant was transferred to a 2-mL glass sample vial and analyzed on a UPLC-MS/MS system (Waters Corp., Milford, MA).
Quantitation Curve 101061 Both reference standards and stable isotope-labeled standards were dissolved in appropriate solvents as indicated below. As the quantitation curve range is different for each compound and the concentration of each metabolite present in real samples varies, the pooled sample was used to estimate the quantitation range. The designated concentrations for each compound/metabolite were obtained. The calibration/linearity equation and corresponding regression coefficients (R2) were calculated using the QuanLynx Application Manager (Waters Corp., Milford, MA) and the limit of quantitation was defined as the lowest concentration in the calibration curve in this study. The calibration curves generated in these experiments for biomarkers cysteine, malate, uracil, N-acetylaspartate, x.anthine, alanine, betaine, and proline are shown in Figures 1 to 8. Figure 9 contains all of the actual biomarker measurements from the various tissue samples in this set of experiments. Figure 10 demonstrates that the biomarkers of the present invention are capable of being quantitated because the measured value of a particular biomarker in the tissue fell within the range of the calibration curve for that biomarker. In the figure, the boxes in gray indicate the range on the calibration curve where the quantity of biomarker in the tissue samples fell. The numerical values in the figure are the actual values of the calibration curves.
101071 Several mobile phases were compared during these experiments including I) A:
acetonitrile (0.2% formic acid, pH = 3), and B: water (0.2% formic acid, pH =
3); II) A:
acetonitrile, and B: water; III) A: acetonitrile (5 mM ammonium acetate, pH =
5.5), and B:
water (5 mM ammonium acetate, pH = 6); IV) A: acetonitrile (5 mM ammonium acetate, pH =
3.8), and B: water (5 mM ammonium acetate, pH = 4); V) A: acetonitrile (5 mM
ammonium acetate, pH = 3), and B: water (5 mM ammonium acetate, pH = 3). Based on chromatographic performance and overall sensitivity, the mobile phase I was finally selected.
The elution gradient was optimized with the goal of separating as many as possible of the targeted compounds without significantly increasing analytical time.
101081 Different reconstitution solvents were compared to maximize the extraction efficiency without compromising the chromatographic performance. A total of five representative solvents were used including I) acetonitrile/methanol = 75:25 (v/v); II) acetonitrile/water = 80:20 (v/v); III) acetonitrile/water = 65:35 (v/v); IV) acetonitrile/water =
50:50 (v/v); V) water. The optimum dilution and reconstitution solvent was acetonitrile/water =
50:50 (v/v) in terms of peak shapes and recovery. A maximal and consistent recovery was achieved by a two-step reconstitution with 100 }IL of acetonitrile followed by 100 1.tL of water.
101091 A UPLC-MS/MS system (ACQUITY 1JPI.,C-Quattro Premier XE M.S, Waters Corp., Milford, MA) was used. The system was operated in electrospray ionization (ESI) positive mode. The optimized instrument settings are briefly described in Table la-b. (can probably leave as table but have to renumber) Gradient solvent B = 0.2% formic acid in water and gradient solvent A = 0.2% formic acid in acetonitrile.
101101 Tables la and lb below show the instrument settings for this set of experiments:
Table la. UPLC-MS/MS instrument settings 13)11.02iMERgagagagagagagagagagag:
1.7 iM Van-GuarO pic-colimin (2.1x 5 nun) and Column ACQUITY UPLC BE) l I IILIC 1.7 !AM analytical column (2.1 x 100 mm) Column Temp (*C) 40th 0.5 Sample Manager Temp ( C) 4 0.5 0-1 min (5% B), 1-2.5 min (5-10% B), 2.5-5 mm (10-20% B), 5-7 min (20-(31-adieu) Conditions 100%13), 7-8 min (100% B), 8-8.2 min (100-5% B), 8.2-9 min (5% B).
Flow Rate (mUrnin) 0.40 Capillary (kV) 4.0 Sampling Cone (V) See Table lb ibr details Collision Energy See Table lb for details Extraction Cone (V) 4.0 Source Temp ( C) 120 Desolvation Temp (CC.) 350 Desolvation Gas Flow 1000 Cone Gas (L/Hr) 50 Table lb. M.S/M.S parameters for compound detection Compound Multiple reaction monitor ' Cone Collision (MRM) transition voltage energy ala3nine 90 >44 30 10 alanine-C13 91 >45 25 10 uracil 113 > 96 30 15 uracil-d4 115 > 98 35 20 proline 116 > 70 35 10 L-proline- 2,5,5-d3 119 > 73 40 15 cysteine 122 > 76 20 10 Cysteine-C13 123 >76 25 15 betaine 118 > 59 20 15 betaine-trimethyl-d5 hydrochloride 127 > 68 35 15 xanthine 153 > 110 30 15 xanthine 1,3-N15 155> 111 35 20 N-acetylaspartate 176> 134 20 10 N-acetyl-aspartate 2,3,3-d3 179> 137 20 10 malate 133 > 71a 25 15 malate 2,3,3-d3 136:> 117a 25 15 a These compounds were detected in negative mode.
101111 Using UPLC-MS/MS with MRM mode, the following metabolites were within the concentration range: betaine, malate, proline, uracil, xanthine, cysteine, and alanine. Although the concentration of N-acetylaspartate in the test sample was on the edge of the LOQ, it is still a viable biomarker because further refinement of the quantitation methods cause it to be within the linear range of the curve (see Figure 10).
101121 This first set of experiments (above) was used to determine which metabolites could be quantitated such that they could be viable biomarkers for prostate cancer. The next set of experiments (below) confirmed these metabolites as biomarkers for prostate cancer.
Identification of Prostate Cancer Bioinarkers 10113] Single core needle biopsies obtained ex vivo from surgical prostatectomy specimens were immediately placed in 80% aqueous alcohol and transferred to formalin after 12-24 hours.
The alcohol was retained, dried down and the residue reconstituted with 95 tiL
of acetonitiile plus 95 !IL of water. Histology was performed on exactly the same tissue.

Standards and solvents 101141 Reference standards and stable isotope-labeled standards were purchased from Sigma-Aldrich (St. Louis, MO) including betaine, malate, proline, N-acetylaspartate, N-acetylglucosamine, uracil, xanthine, cysteine, alanine. Stable isotope-labeled chemicals including glucosamine CI3 dydrochloride, betaine-trimethyl-d5 hydrochloride, xanthine 1,3-N15, cysteine C13, malate-2,3,3-d3, and L-proline 2,5,5-d3 were obtained from Cambridge Isotope Laboratories Inc. (Andover, MA) and uracil-d4, N-acetyl-aspartate 2,3,3-d3, and alanine-C13 were from C/D/N ISOTOPES INC. (Quebec, Canada). Chemicals including methanol (Optima LC-MS), acetonitrile (Optima LC-MS) and formic acid (Optima LC-MS) were purchased from Thermo Fisher Scientific (FairLawn, NJ). Ammonium acetate and acetic acid (Glacial) were purchased from Sigma-Aldrich. Sodium formate solution (0.05 M NaOH
0.5% formic acid in 90:10 2-propanol: water, Waters Corp., Milford, MA) was used for instrument tuning and calibration. Ultrapure water was produced by a Mill-Q
Reference system equipped with a LC-MS Pak filter (Millipore, Billerica, MA).
Source of Samples 101151 A total of 30 study samples extracted from prostate needle biopsies were provided and stored at 4 C prior to sample preparation and analysis. The sample (CA5661_1) was not analyzed due to instrument failure (over-pressurization) during injection. The actual number of samples analyzed was 29. The tissue samples from 12 patients used to quantitate the biomarkers were as follows: 15 Tumor samples (1 sample pT3a and 14 samples pT2) and 14 Paired adjacent non-tumor sample.
Sample preparation 101161 Each 1.9 mL of sample was transferred to a 5raL glass vial and dried under a gentle nitrogen flow at room temperature (Glas-Col Nitrogen Evaporator System, Terre Haute, IN).
The residue was reconstituted with 95 Lut of acetonitrile plus 95 1.tL of water. The mixture was centrifuged at 14,500 rpm for 20 mm (Microfuge 22R centrifuge, Beckman Coulter, Inc., Atlanta, GA). A 150 lit aliquot of the resulting supernatant was transferred to 2 mL glass sample vial and analyzed on a UPLC-MS/MS system (Waters Corp., Milford, MA).
Quantitation curve 101171 Both reference standards and stable isotope-labeled standards were dissolved in appropriate solvents (methanol or water) based on their solubility. The stable isotope-labeled standards were used as internal standards for their corresponding analytes, and thus were used to compensate for possible variations during sample preparation, injection, chromatography, matrix effects, etc. The quantitation curve solutions were prepared by mixing each aliquot of reference standard stock solution followed by a series of dilution with a mixture of methanol and water (50:50, v/v). The designated concentrations for each compound were obtained. The calibration equation and corresponding regression coefficients (R2) were calculated using the QuanLynx Application Manager (Waters Corp., Milford, MA) and the limit of quantitation was defined as the lowest concentration in the calibration curve. These curves are shown in figures 13-21.
101181 mPREF samples including tumor/non-tumor were analyzed using the developed assay method described above and the concentration ranges obtained. These metabolites were assayed on a single LC/MS run.
Tables 2a and 2b below show the instrument settings for this set of experiments:
Table 2a. UPLC-MS/MS instrument settings UPLC
AcQurry UPLC BEH HILIC 1.7 AM VanGuard pre-column (2.1 x5 mm) and Column ACQUITY UPLC BEH HILIC 1.7 M analytical column (2.1 x 100 mm) Column Temp ( C) 40 :1: 0.5 Sample Manager Temp ( C) 4 0.5 0-1 min (5% B), 1-2.5 min (5-10% B), 2.5-5 min (10-20% B), 5-7 min (20.
GradientConditions 100% B), 7-8 min (100% B), 8-8.2 min (100-5% B), 8.2-9 min (5% B) Flow Rate (mLimin) 0.40 Quattre XE Premier MS
Capillary (kV) 4.0 Sampling Cone (V) See Table 2b for details Collision Energy See Table 2b for details Extraction Cone (V) 4.0 Source Tem2( C) 120 Desolvation Temp ( C) 350 Desolvation Gas Flow 1000 (L/Hr) Cone Gas (L/Hr) 50 Table 2b. MS/MS parameters for compound detection Compound Multiple reaction monitor Cone Collision (MRM) transition voltage energy (V) or) alanine 90 > 44 30 10 alartine-C13 91 >45 25 10 uracil 113 > 96 30 15 uracil-d4 115 > 98 35 20 proline 116 > 70 35 10 L-proline-2,5,5-d3 119> 73 40 15 cysteine 122 > 76 20 10 cysteine-C13 123> 76 25 15 betaine 118> 59 20 15 betaine-trimethyl-d5 hydrochloride 127 > 68 35 15 xanthine 153 > 110 30 15 xauthine1,3-N15 155 > 111 35 20 N-acetylaspartate 176> 134 20 10 N-acetyl-aspartate 2,3,3-d3 179> 137 20 10 malate" 133 > 71 25 15 malate 2,3,3-d3 136 > 117 25 15 N-acetylglucosatnine 222> 138 20 15 glucosamine-C13 dydrochloride 181 > 73 30 15 ' These compounds were detected in negative mode.
101191 Figures 13 to 21 are the calibration curves for each of the biomarkers analyzed in these experiments. Figure 11 contains the quantitation data for the biomarkers of the present invention in this set of experiments. Figure 12 demonstrates that the quantitation range for each biomarker (represented by the grey band) falls within the linear portion of the calibration curve for the biomarkers. Therefore, each of these biomarkers can be quantitated in an 18 gauge core biopsy of prostate tissue.
111. Normalization of biomarkers to tumor surface area 101201 Matphometric determination of the amount of tumor, non-tumor, glands and stroma in a given biopsy was used to determine that the biomarkers of the present invention correspond with the amount of tumor present in a biopsy. The data was normalized to the total biopsy surface area for each biopsy core. Biopsy surface area was obtained by scanning glass slides on a high resolution flatbed scanner. Dark pixels were converted to surface area using the program "Imager. The resulting surface area was then used to convert data to p1/1/cm2.
The uM/cm2 values for samples without tumor and with tumor are provided below in Table 3.
Table 3 Biomarker No Tumor No Tumor Tumor Lo Std Hi Std Tumor uM Tumor uM/cm2 Corrected uM uM
uM uM/cm2 uM
betaine 0.079 0.189 2.041 4.712 0.46 0.226 231 malate 1.017 1.248 30.233 33.92 3.47 1.25 1281 proline 1.472 1.763 42.943 47.543 4.99 0.262 269 N-acetylaspartate 0.171 0.153 2.794 2.992 0.312 1.11 1133 Mei! 1.443 2.030 41.127 50.555 5.15 3.74 3830 , xanthine 0.127 0.154 3.898 4.377 0.766 0.0833 85.3 cysteine 28.39 44.71 877.901 1209.76 127.94 1.46 1497 alanine 31.51 _____ 35.27 948.749 927.435 113.7 0.835 855 N- 0.698 1.195 20.859 28.970 2.57 0.387 396 acetylglucosamine 101211 In table 3 above, No Tumor uM is the raw data; uM/Cm2 is the data normalized to surface area of the biopsy core; Lo Std/Hi Std are the low and high end of the standard curves.
The calculation of the "Tumor Corrected" data is described below.
101221 The data was also normalized based upon percent surface area of tumor. Percent tumor was assessed by microscopic examination of the biopsies by a pathologist. Raw data for each biomarker was converted to the equivalent of 100% tumor by expressing percent in decimal form and multiplying by the appropriate factor to equal one (e.g. 10%
= 0.1; 0.1 x 10=
1). This is referred to as "tumor corrected" in Table 3. The no tumor values were then compared to the tumor corrected values to demonstrate the difference between the amount of the biomarker in normal tissue and the amount in tumorous tissue. Figure 23 shows the comparison of the uracil, N-acetylaspartate, proline, xanthine, betaine, malate, and N-acetylglucosamine biomarkers in normal tissue (black) versus tumor tissue (gray). Figure 24 shows the comparison of the cysteine and alanine levels in normal tissue (black) versus tumor (gray). These data indicate that the biomarkers of the present invention can be used to quantitatively determine the amount of cancer tissue in a particular prostate biopsy. Therefore, these biomarkers can be used to detect prostate cancer, determine prostate cancer prognosis, monitor the treatment of the disease, and screen possible new treatments for prostate cancer.
101231 While the invention has been illustrated and described in the figures and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. In addition, all references and patents cited herein are indicative of the level of skill in the art and hereby incorporated by reference in their entirety.
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Claims (27)

1. A method for screening for prostate cancer in a subject comprising the steps of:
(a) providing a biological sample from a subject;
(b) detecting at least one biomarker in said sample, said biomarker selected from the group consisting of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, anti N-acetylglucosamine; and (c) correlating said detection with a status of prostate cancer or no prostate cancer.

2. A. method for screening for prostate cancer in a subject comprising the steps of:
(a) providing a biological sample from a subject;
(b) detecting at least one biomarker in said sample, said biomarker selected from the group consisting of betaine, malate, proline, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine; and (c) correlating said detection with a status of prostate cancer or no prostate cancer.

3. The method of claim 1 wherein said detecting at least one biomarker is performed by mass spectrometry.

4. The method of claim 1, wherein the biological sample is selected from the group consisting of biological fluid and tissue.

5. The method according to claim 4, wherein the biological fluid is whole blood, serum, plasma, or urine.

6. The method according to claim 4, wherein the tissue is a prostate tissue sample.

7. The method of claim 1, wherein the biological sample is contacted with a solvent capable of extracting the at least one biomarker.

8. The method according to claim 7, wherein the solvent is methanol or ethanol.

9. A method of diagnosing prostate cancer in a subject, comprising the steps of:
(a) obtaining one or more test samples from a subject;

(b) detecting at least one biomarker in the one or more test samples, wherein the biomarker is selected from: betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine;
(c) quantitating the amount of the at least one biomarker; and (d) correlating the quantitation of the at least one biomarker with a diagnosis of prostate cancer.

10. -- A method of diagnosing prostate cancer in a subject, comprising the steps of:
(a) obtaining one or more test samples from a subject;
(b) detecting at least one biomarker in the one or more test samples, wherein the biomarker is selected from: betaine, malate, proline, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine;
(c) quantitating the amount of the at least one biomarker; and (d) correlating the quantitation of the at least one biomarker with a diagnosis of prostate cancer, wherein the correlation takes into account the amount of the at least one biomarker in the one or more test samples compared to a control amount of the at least one biomarker.

11. -- The method of claim 10 wherein the correlation takes into account the amount of the at least one biomarker in the one or more test samples compared to a control amount of the at least one biomarker.

12. -- The method of claim 10 wherein the test sample is selected from the group consisting of urine, whole blood, serum, plasma, and prostate tissue.

13. -- A. method of monitoring the effect of a prostate cancer drug or therapy on a subject comprising:
(a) providing a biological sample from the subject;
(b) contacting the biological sample with a solvent capable of extracting at least one prostate cancer biomarker selected from the group consisting of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine;
(c) quantitating the amount of the at least one prostate cancer biomarker;
(d) providing the subject with an anti-prostate cancer drug or therapy;

(e) quantitating the amount of the at least one prostate cancer biomarker using steps (a) and (b); and (f) corrlating the two measurements with a diagnosis that the prostate cancer is regressing or progressing.

14. A multiplexed assay for screening for prostate cancer in a subject comprising the steps of:
(a) providing a biological sample from a subject;
(b) quantitating at least two or more biomarkers in said sample, said biomarkers selected from the group consisting of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine;
(c) correlating said quantitation with a status of prostate cancer or no prostate cancer.

15. The method of claim 14 wherein said quantitating at least two or more biomarkers is performed by liquid chromatography in tandem with mass spectrometry.

16. The method of claim 14, wherein the biological sample is selected from the group consisting of biological fluid and tissue.

17. The method according to claim 16, wherein the biological fluid is whole blood, serum, plasma, or urine.

18. The method according to claim 16, wherein the tissue is a prostate tissue sample.

19. The method according to claim 14, wherein the biomarkers betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine are quantitated in the same assay.

20. The method according to claim 14, wherein the biomarkers betaine, malate, proline, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine are quantitated in the same assay.

21. A multiplexed method for detecting prostate cancer in a subject comprising the steps of:
(a) providing a biological sample from the subject;

(b) contacting the biological sample with a solvent capable of extracting two or more prostate cancer biomarkers selected from the group consisting of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine;
(c) quantitating the amount of the two or more biomarkers present in the biological sample; and (d) correlating the amount of the two or more biomarkers with the presence or absence of prostate cancer.

22. The method of claim 21, wherein the quantitating differentiates between different stages of prostate cancer.

23. The method of claim 21, wherein the quantitating is part of a diagnosis or prognosis of prostate cancer in the subject.

24. The method according to claim 21, wherein the solvent is methanol or ethanol.

25. The method of claim 21, further comprising the step of performing additional histological analysis on the extracted biological sample.

26. A method of diagnosing prostate cancer in a subject, comprising the steps of:
(a) obtaining one or more test samples from a subject;
(b) detecting at least one biomarker in the one or more test samples, wherein the biomarker is selected from the group consisting of betaine, malate, proline, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine;
(c) quantitating the amount of the at least one biomarker;
(d) determining the Gleason score of the one or more test samples;
(e) correlating the quantitation of the at least one biomarker and the Gleason score with a relative risk of T2 versus T3 prostate cancer.

27. A kit for diagnosing prostate cancer in a subject comprising:
(a) a vial for collecting a biological sample from the subject;
(b) a solvent for extacting biomarkers from the biological sample, the biomarkers selected from the group consisting of betaine, malate, proline, N-acetylaspartate, uracil, xanthine, cysteine, alanine, and N-acetylglucosamine;

(c) instructions for performing the extraction of the biomarkers;
(d) instructions for quantitating one or more of the biomarkers;
(f) instructions for correlating the quantitation of the one or more biomarkers to a diagnosis of prostate cancer or normal.